University Of California Santa Cruz

NIH Research Projects · FY 2026 · 2023-12

Project Summary/Abstract Telomere length plays a pivotal role in cancer and age-related degenerative disease. Telomeres are made of simple tandem repeats, added by the enzyme telomerase, to establish a length distribution. A mechanistic understanding of the forces that regulate telomere length is needed to develop approaches to disease. Current models for how the telomere length equilibrium is maintained suggest that ends with fewer telomere repeats are more frequently elongated than ends with many repeats implying that all telomeres are regulated around the same length distribution. We developed a nanopore sequencing method to measure telomere length in yeast at the nucleotide level. Surprisingly, we found that each telomere had a distinct length distribution that was maintained over hundreds of cell divisions. This discovery requires that current models for length regulation be revised. In this proposal we detail specific experiments to test mechanisms that may modify the length distribution at specific chromosome ends: 1. Subtelomeric sequences 2. Telomere transcription/telomere RNA (TERRA), and the Sir2/3/4 histone modification complex. We will use computational approaches to determine if specific sequence motifs are associated with long or short telomeres, and we will systematically delete DNA binding sites to test their role. We will use nanopore RNA sequencing to study the long non-coding RNA TERRA, and test whether the RNA, or the action of transcribing the telomere play a role in end-specific telomere lengths. Finally, we will test the role of the histone deacetylase Sir2 and the interaction of Sir4 with Ku in determining end-specific telomere lengths. These experiments will implicate or rule out the specific factors that establish different length distributions. Understanding molecular mechanism(s) that influence telomere length distributions will set the stage to specifically manipulate telomere length. Because mechanisms of telomere length regulation are conserved across species, our experiments will set the stage for approaches to alter telomere length to treat human disease.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY Influenza virus infection triggers lung inflammation and pathology and is a leading cause of acute respiratory distress syndrome. Following viral clearance, lung inflammation and pathology can persist preventing recovery from severe cases of infection. Such persistent lung inflammation and pathology, also referred to as Post-viral Lung Disease (PVLD), is an underappreciated aspect of influenza virus infections, even though many patients suffer extended hospital stays, and 25 – 50% of patients continue to experience respiratory symptoms for at least 2 months after hospital discharge. Severe influenza virus infections also exacerbate other chronic respiratory diseases, including COPD and idiopathic pulmonary fibrosis. Currently, there are no approved pharmacologic interventions to improve recovery after viral clearance and to prevent or reverse PVLD and its long-term effects. PVLD is characterized by the persistence of inflammatory immune cells in the lung tissue and the failure of effective alveolar epithelial repair capable of restoring tissue function. The cellular and/or molecular mechanisms driving the persistence of inflammatory immune cells and preventing effective alveolar repair in the absence of ongoing viral replication are not known. We recently identified lung fibroblasts as drivers of inflammation and influenza disease severity. We showed that a lineage of lung fibroblasts, which we term inflammatory fibroblasts (iFibs), are especially important for driving pathogenesis, and not only promote hyperinflammation, but also prevent restoration of normal alveolar function during acute infections. Our preliminary data now demonstrate that these iFibs do not die during the acute phase of the infection, but instead persist into post-viral stages of influenza disease. Together, these data allow us to hypothesize that a subset of lung fibroblasts survives regulated cell death during acute IAV infection, differentiate into activated iFibs, persist in damaged lung tissue well after viral clearance, and drive PVLD by continued production of inflammatory mediators. We will test this hypothesis by addressing the following three key questions: (1) From which cells, and when, do PVLD-driving inflammatory fibroblasts arise? (2) Do inflammatory fibroblasts persist because they do not undergo regulated cell death, and (3) What is the therapeutic potential of inhibiting persistent inflammatory fibroblast activity in PVLD? Successful completion of this project will identify a distinct lineage of inflammatory lung fibroblasts as the cell type that initiates and drives PVLD and the mechanism of their persistence following infection. This research will identify novel therapeutic entry points to prevent or reduce PVLD before it develops to the point that it becomes irreversible.

NIH Research Projects · FY 2025 · 2023-09

Project Summary Mobility, orientation, and spatial awareness can be challenging for people who are blind. Navigating new or unfamiliar environments without sight can be confusing and stressful, and potentially dangerous. As a consequence, it has been reported that at least 30% of blind individuals do not make independent trips outside of their home. For sighted and blind travelers alike, maps represent a valuable tool for spatial cognition. Maps can be made accessible to blind users using tactile format, or as multimodal vibrational/audio maps (a format pioneered by co-I Giudice, himself a blind individual). Research has shown that, when given the opportunity to explore an accessible map beforehand (pre-journey learning), blind travelers may achieve better spatial awareness of locations, which may help with maintaining orientation and determining new routes. Tactile maps are traditionally created by professionals, often following “tricks of the trade” and using non-standardized symbols. Recently, there has been a push towards systematic approaches to tactile map creation that would enable wider diffusion and consistent spatial symbols rendering. Online services (e.g., TMAPS) have been created, that allow anyone to specify a certain region to be mapped, and have a tactile map automatically generated for that region, sourcing from an open access geographical information system. Existing systems for on-demand map creation, however, can only map outdoor areas. There is no such service for building indoors, and in fact, very little research work has addressed the systematic production of tactile indoor maps. Indoor environments are generally less structured than typical urban layouts. Moving about in a building may require spatial knowledge at multiple scales (from the general building structure to the internal layout of a room). And, unlike widely available GIS data sets, obtaining detailed floor plans in a format amenable for tactile map creation can be challenging. In this application, we propose research and development focusing on a system for the semi-automatic creation of accessible maps (in tactile and vibrational/audio format) of indoor environments. This work will stem from a prior web application (SIM, developed by PI Manduchi’s team) that allows anyone to trace an existing floor plan to generate an embossable map. Our research activities will be organized around the following Specific Aims: Facilitating acquisition and “vectorization” of floor plans (Aim 1); Automatic conversion of a vectorized map (expressed in terms of spatial primitives) to an accessible map at any desired scale, by means of cartographic generalization operators and carefully designed symbol sets (Aim 2); User studies with blind subjects, designed to evaluate the effectiveness of the maps produced by our new SIM system (Aim 3); Transferring the new SIM web app technology to our partner institution, the Vista Center for the Blind of Palo Alto, which will make it available as a service to interested stakeholders.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Next generation sequencing technologies have greatly expanded the size of the known transcriptome. Many newly discovered transcripts are classified as long noncoding RNAs (lncRNAs) which are assumed to influence phenotype through sequence and structure and not via translated protein products, despite the vast majority of them harboring short open reading frames (sORFs). Recent advances have demonstrated that the noncoding designation is incorrect in many cases and that sORF-encoded peptides (SEPs also called micropeptides) translated from these transcripts are important contributors to diverse biological processes including inflammation and cell viability. An appropriate inflammatory response is critical for host defense against pathogens, but chronic inflammation is associated with many diseases. Macrophages play a significant role in both initiating and resolving inflammation and understanding their part in this process is scientifically and practically important. One long studied - yet not fully understood - model of macrophage proinflammatory polarization involves lipopolysaccharide (LPS) activation of toll-like receptor 4 (TLR4). Following detection of LPS, a signaling cascade initiates leading to the translocation of transcription factor NFkB to the nucleus. This is followed by increased expression of established inflammatory cytokine and interferon genes. However, this also results in changes in expression of many unstudied lncRNAs. In addition to changes in transcription, changes in translation also follow inflammatory stimulation and these alterations have been observed to increase translation of “noncoding” regions in some cases. Indeed our lab and others have observed dramatic changes in associations between lncRNAs and polysomes following LPS stimulation in mouse macrophages. Therefore, the central hypothesis of this proposal is that translation of lncRNAs produce SEPs that play important roles in the TLR4-NFkB inflammatory response and in macrophage viability. To test this hypothesis, I present a strategy for screening lncRNA sORFs with evidence of coding potential in mouse macrophages. The screen will make use of macrophage cell lines with a NFkB-GFP reporter and CRISPR- Cas9 casssette. Secondly, I propose a biomolecular pipeline for mechanistically characterizing a selection of novel SEPs. This work has the potential to identify many novel SEPs that are important for regulating the inflammatory response. This would further our understanding of a model inflammatory pathway and could help identify novel peptides with therapeutic potential or as therapeutic targets.

NIH Research Projects · FY 2026 · 2023-07

SUMMARY Each adult human harbors hundreds of bacterial species in their intestine. However, the networks of microbe- microbe interactions that underly the stable co-existence of resident species, and exclude additional species, are not well defined. The intestinal lumen is a turbulent, semi-fluid landscape where microbial cells and dietary plant cell wall fragments are distributed with high heterogeneity, and redistributed on the time scale of seconds. We propose that bacteria selectively adhere to dietary particles in the gut lumen and that interactions with their co-adherent microbial partners dictate whether they persist. We created multiplex libraries of artificial food particles (consisting of glycan-coated magnetic beads) to measure gut bacterial adhesion in vivo, and discovered that many members of the phylum Bacteroidetes adhere to dietary glycan particles in a strain- specific and glycan-specific manner. We will first identify families of adhesion proteins required for these binding phenotypes using transposon mutagenesis and insertion site sequencing. Next, we will identify networks of interacting strains that co-adhere to dietary particles. Using orally administered libraries of fluorescently labeled beads, we will map co-adhesion networks in gnotobiotic mice colonized with strains that have evolved together in a single donor host. Finally, we will establish a mutational selection strategy that permits the simultaneous generation of different binding specificities in genetically intractable gut microbes. Analysis of these mutations will reveal the potential origins of adhesion-dependent interspecies relationships. These studies will shed light on the poorly defined spatial structure of the gut microbiota. The technologies we develop in this proposal hold promise as a means to intentionally position the members of microbial communities in physical configurations that prevent or ameliorate metabolic, immunologic, and infectious diseases.

NIH Research Projects · FY 2024 · 2023-06

Project Summary (Max 30 lines) From early childhood, children need to develop views about when to help others and when to refrain. In deciding whether to help, children often need to balance their personal concerns with their own interests against their moral concerns with the interests of a recipient. Children who never help others may become socially isolated, whereas children who always help others may be taken advantage of. Developing a discerning prosociality is therefore key to healthy development. The preschool years is a transformative period in prosocial development, when children’s household involvement increases in many communities and when they become more able to reason about competing moral and personal considerations. However, much prior research on prosocial development has examined stable individual differences, seeking to identify characteristics of children who, on average, help more than others. In contrast, much work on children’s moral reasoning and judgments has focused on situational variability—how children judge helping as okay in some situations but not others—rather than stable individual differences. The proposed research will test predictions of a model that incorporates insights from both of these research traditions by examining how individual stability and situational variability in evaluative reasoning about helping can explain stability and variability in prosocial behaviors. To explain how a discerning prosociality develops, the proposed model also bridges a second tension in the field: that between caregiver socialization and child autonomy. Research on caregiver socialization has often defined healthy development as consisting of children adopting the values and practices of their caregivers. By contrast, constructivist approaches have focused on how children scrutinize the values and practices of their caregivers, accepting some and rejecting others. According to the integrative model tested by the proposed research, children’s evaluative reasoning about helping develops through conversations with caregivers, who can draw children’s attention to either personal or moral aspects of the helping situation. To test key model predictions, the proposed research will involve naturalistic observations, structured interviews, and storybook conversations. Through these activities, the project will assess caregiver-child interactions around helping, as well as children’s prosocial reasoning, judgments, and actions in response to both hypothetical and actual events. An ethnically diverse sample of 150 4- to 6-year-olds and their families will be recruited to participate in one home visit and one virtual session. This initial study will test predictions of the basic scientific model about how preschoolers develop prosociality through reasoning. The findings will inform a larger intervention study aimed at leveraging everyday caregiver- child conversations to strengthen children’s healthy prosociality.

NIH Research Projects · FY 2025 · 2023-06

úú PROJECT SUMMARY/ABSTRACT Delivering diagnostic services at the point-of-care (POC) can improve the quality of healthcare in clinics, in emergency settings, or at home, which can potentially ease hospitals’ burden, for instance, during the COVID- 19 pandemic. Precision and personalized medicine revolution also require POC testing to provide readily available biomarker information to clinicians. The goal of this career development proposal is to create fast, inexpensive, sensitive, and reliable molecular diagnostics to address the 21st-century healthcare challenges. The central hypothesis is that we can efficiently utilize computational protein design to create modular allosteric protein switches, named LOCKR (Latching Orthogonal Cage–Key pRotein), that enable the rapid and reversible conformational changes upon interaction. As a proof of principle, we demonstrate that LOCKR- based biosensors can be configured to produce bioluminescence upon the addition of clinical targets (e.g., botulinum toxin, cardiac troponin I, HER2 receptor, Fc domain, anti-HBV mAb, anti-SARS-CoV2 antibodies, and SARS-CoV2 receptor-binding domain/spike protein, Fig 1 and 2) in homogeneous “all-in-solution” assays. Due to the modularity of LOCKR sensor platform and the advance in de novo binder design for arbitrary protein targets, we proposed the integration of both features as the universal strategy to develop tailored biosensors for user-defined targets. The main specific aims for the independent phase are to iteratively expand LOCKR-based diagnostics with the synergy of (1) de novo protein binder design to directly detect various disease protein biomarkers, and (2) indirectly detect the antibodies that compete with the designed interface, as POC devices; and (3) to repurpose the original luminescence signal with other compatible readouts by exchanging the reporter modules. For more specific proof-of-concept projects during the mentored phase, I describe in Aim 1 the use of state-of-the-art computational protein design methods to create an interleukin-6 binder and biosensor. In Aim 2, I propose a general way to develop antibody biosensors by demonstrating COVID-19 serological tests as an example. With my expertise in biosensor engineering, I attempt in Aim 3 to develop a ratiometric bioluminescence resonance energy transfer (BRET) biosensor to analyze the HBV antibody and a colorimetric biosensor to measure human cardiac troponin I level. Ultimately, I anticipate this new sensor platform is significant for the development of robust protein sensors that will be broadly applicable to arbitrary targets and enabling its POC compatible readouts for future diagnostics.

NIH Research Projects · FY 2026 · 2023-06

Project Summary Cortico-cortical projection neurons (CCPNs) connect cortical areas with each other to facilitate sensory processing and execute appropriate motor actions. Defects in intra-cortical connectivity are associated with a variety of neural circuit disorders such as dyslexia, autism, and schizophrenia. Because these diseases have genetic components and potentially arise from altered brain development, it is important to understand how CCPNs know which area to target and which synaptic inputs to receive. The long-term goal of my research program is to gain mechanistic insights into cortical circuit assembly at the single cell level in an effort to understand underpinnings of neurodevelopmental disorders and develop new therapies. In doing so, we will be able to identify molecular and genetic mechanisms that link gene expression and neural activity to neuronal connectivity. The objective of this proposal is to identify mechanisms by which long-range cortico-cortical neuronal connectivity is established in the mammalian cortex using the mouse visual system as a model. Our central hypothesis is that V1 neurons, projecting to the AL (anterolateral: V1→AL) or the PM (posteromedial: V1→PM) higher visual areas, differ in timing and molecular regulation of their axonal projection development, and their input versus output connectivity is specified by two distinct rules: early specification and synaptic pruning mechanism, respectively. We will test this hypothesis with the following aims: 1) we will determine the patterns and timing of cortico-cortical neuronal projection development in the mouse visual cortex. 2) we will determine the roles of Teneurins, cell-adhesion molecules, in specifying the projection identities of V1→AL and V1→PM neurons. 3) we will determine developmental principles of ‘like-to-like’ cortico-cortical feedback circuit formation. This research is significant because elucidating the developmental mechanisms of neural circuit assembly will provide cell-type or circuit-specific therapeutic interventions for specific aspects of neurodevelopmental and psychiatric disorder phenotypes. The proposed research is innovative because we are using and developing the technical solutions to allow us to target gene expression and capture the rapid developmental connectivity dynamics of layer- and projection-specific cortical neurons. Results will provide a comprehensive understanding of how a long-range connectivity network arises at the cellular level. This new knowledge will have a positive impact on the neuroscience field as it will establish a solid foundation to provide connectivity-based therapeutic interventions for neurodevelopmental disorders.

NIH Research Projects · FY 2026 · 2023-05

Metastasis remains the primary cause of death in women with breast cancer. While it is well established that tumor cells are heterogeneous and minority populations within the tumor have tumor-initiating and metastatic capabilities, the identity of metastasis initiating cells (MICs) in human breast cancer remains controversial. Until we can identify these cells and study the molecular pathways regulating them, the development of new therapeutic strategies will be hindered. The long-term goal of this proposal is to understand the intrinsic and extrinsic molecular signaling regulating MICs. Recently, using single-cell RNA sequencing data from breast cancer patient samples and sophisticated computational approaches we identified an immature population of cells in breast cancer that express hematopoietic stem cell transcriptional adaptor and the T-cell oncogene, LMO2. The objective of this proposal is to determine the molecular mechanism of LMO2 in promoting metastasis. We hypothesize that LMO2+ cells are a population of MICs in breast cancer that are activated in response to inflammation and LMO2 is a key adaptor that regulates this process. Our hypothesis is based on our preliminary results that demonstrate that Lmo2+ cells are metastatic, predict poor distant recurrence-free survival in patients, LMO2 knockdown reduces metastasis in multiple human tumor models, and LMO2 is required for STAT3 activation in response to IL6 and TNFα. The rationale underlying this proposal is that αidentifying the signaling of LMO2 in metastasis will elucidate targets in MICs that are open to therapeutic intervention. Guided by strong preliminary data, our proposal will 1. Determine whether LMO2 is required for inflammation-induced metastasis. 2. Determine the detailed molecular signaling regulated by LMO2-STAT3 axis. 3. Determine whether LMO2+ cells utilize vascular mimicry to metastasize. The proposed research is significant because it will enable the design of therapeutic strategies targeting MICs in breast cancer. Previous research studies on the identification of MICs have relied on established lineage markers. The proposed research is innovative because we are focusing on a population of LMO2+ MICs that were agnostically identified from single-cell RNA sequencing in breast cancer patient samples. The proposed research will substantially enhance our understanding of MICs and lay the foundation for novel strategies to treat metastatic breast cancer.

NIH Research Projects · FY 2025 · 2023-05

ABSTRACT Our team proposes to lead the SSPsyGene consortium into the Data Biosphere. We will do this by adapting data biosphere technology and management techniques we have already deployed for other NIH institutes, NIH Common Fund, the NIH Office of the Director, the Chan Zuckerberg Initiative (CZI), and the California Institute for Regenerative Medicine (CIRM), making SSPsyGene interoperable across multiple disease areas. We also bring our expertise with neurological data through our involvement with BICCN, Psychiatric Cell Map Initiative, CZI’s Pediatric Brain Map, NHGRI's Center for Live Cell Genomics/Biotechnology, and our close relationship with PsychENCODE and the Allen Brain Institute. For SSPsyGene, we have 4 major tasks: (1) We will assemble all the information necessary to empower the consortium to choose between 100 and 250 genes to experimentally characterize (Aim 2). We have identified more than 20 different types of information to be integrated for this purpose, many of which are already in the UCSC Genome Browser. We will apply multiple ranking algorithms to this integrated information source to guide the SSPsyGene Consortium’s decision process. (2) We will work to establish an ontology structure that is sufficiently expressive yet fully maintainable, supporting FAIR data use by both researchers and machines (Aim 3). Our previous work with the UCSC Genome Browser and our close relationships with ontology organizations will help us to bridge the gaps between molecular, cellular, tissue/organoid, and model organism measurements, and to extend these resources when needed. Inspired by our experience with the clinical ontologies in OMOP and FHIR, we propose a novel service to allow researchers to query phenotype-phenotype associations in large clinical cohorts, such as All of Us and HEDIS, the database of records from Medicare and Medicaid. (3) We will create a state-of-the-art SSPsyGene Data Biosphere fully compatible with those we created for other NIH institutes (Aim 4). Our emphasis will be on standardization of the data submission process with extensive quality monitoring to ensure timely and effective data release. We will leverage our deep involvement with the Global Alliance for Genomics and Health to ensure all data and metadata will meet FAIR standards. We have experience with the complex data types that will be generated by the SSPsyGene consortium, including -omics, imaging, electrophysiology and other data types. (4) We have served as trusted third party organizers to many NIH consortia, developing a reputation for fairness and impartiality in data sharing and publication, and expertise in coordinating, generating consensus, publishing results, and creating a resource with maximal impact (Aim 5). Based on our strengths in biomedical data, metadata and ontologies, FAIR platforms, and consortium leadership, we are confident that we will achieve all the goals of the SSPsyGene Consortium.

NIH Research Projects · FY 2026 · 2023-01

Macrocyclic peptides (MCPs) can demonstrate antibody-like potency and specificity against "undruggable" targets such as protein-protein interactions. Some MPCs, especially ones found in nature, also have drug-like cell permeability and even oral absorption, leading to the proposition that MCPs define a fertile ground for the discovery of novel, cell permeable inhibitors against undruggable targets. My lab is among the leaders in the worldwide effort to define the factors that govern passive membrane permeability in MCPs. In addition to defining a set of rules for generating molecules in this space, we have shown that existing macrocyclic natural products represent only a tiny fraction of potential permeable scaffolds in this size range (MW 700-1500). As the basic science of membrane permeability in MCPs has continued to mature, new questions have arisen which our lab is uniquely positioned to address: To what extent can side chains sequester polar backbone atoms in the membrane, and, conversely, to what extent can polar side chain functionality be "smuggled" into the membrane via interactions with backbone atoms? Are there scaffold geometries that enhance these effects? What is the fundamental size limit to passive membrane permeability? To what extent can strongly ionizable groups be incorporated into lipophilic MCPs without abrogating permeability? Can DNA-encoded library technology be used to discover novel, membrane permeable scaffolds that greatly enhance the extent to which we can evaluate this chemical space, especially in the higher MW range? Our program will capitalize on recent developments in DNA-encoded library (DEL) technology to generate large (108 - 1012-member) libraries that are diversified at both the side chain and backbone levels. We have shown that DNA-conjugated MCPs can be separated chromatographically based on the permeability of the pendant macrocycle and independent of the encoding DNA molecule, allowing us to use the power of split-pool synthesis and next- generation sequencing to dramatically expand our ability to delineate the constraints on permeability among highly diverse scaffolds well above 1000 MW. Finally, there have been few systematic studies on the impact of scaffold geometry on efflux and hepatic metabolism, which, besides permeability, are important factors that govern pharmacokinetic behavior. We will utilize our powerful split-pool synthesis and MSMS-analytical tool to determine the effect of scaffold geometry on efflux and metabolism, which will further enhance our understanding of this important chemical space. This MIRA proposal seeks to build on a vibrant and successful research program to uncover the basic scientific principles governing drug-like properties in a chemical class that continues to inspire medicinal chemists in their pursuit of ever more challenging targets.

NIH Research Projects · FY 2025 · 2022-09

Project summary: This research project aims to provide a mechanistic and structural model of general and location- dependent eukaryotic elongation factor 2 kinase (eEF2K) regulation and its downstream effects on the ribosome. eEF2K is at the confluence of multiple upstream pathways whose signals it integrates. eEF2K’s only known target is the eukaryotic elongation factor 2 (eEF2), which it phosphorylates on a single site. This eEF2K/eEF2- axis is the predominant regulator of translation elongation and has a general role in cell homeostasis. It is also an essential cue-dependent regulator of protein synthesis in localized regions of specific cell types, for example, after neurotransmitter exposure in the synapses of neurons. eEF2 promotes the translation of specific mRNAs while generally inhibiting translation. The mechanism of this paradoxical phenomenon is entirely unknown. We recently showed that active eEF2K imposes a general translation shutdown in which phosphorylated eEF2 and the phase-separating protein SERBP1 stably bind to ribosomes and renders them idle. This assembly suggests possible mechanisms of eEF2-phosphorylation with respect to ribosome stability, mRNA decay, and ribosome localization that collectively explain how eEF2-phosphorylation might lead to preferential translation of certain mRNAs. eEF2K is associated with numerous human diseases, including neurological dysfunctions, infectious diseases, cancers, and autoimmune disorders. Therapeutics targeting eEF2K are under development but currently lack insufficient specificity. Thus, a mechanistic understanding of eEF2K-regulation and its downstream effects are needed. Under this award, we will pursue two key directions: 1) determine how eEF2K structurally integrates signals from upstream pathways and affects its downstream regulation of eEF2, and 2) determine how phosphorylated eEF2 and idle ribosomes regulate mRNA translation globally and locally. Here, we will test the novel hypotheses that eEF2-phosphorylation regulates ribosome stability, mRNA stability, and ribosome localization, which collectively confers the preferential translation of a specific mRNA subset. We will use an integrated structural biology approach using single-particle and in situ cryogenic electron microscopy, paired with biophysical, biochemical, and cell biology approaches to address these general and location-specific roles of the eukaryotic elongation factor 2 kinase (eEF2K) pathway. Our proposed research program will open the door to promising therapeutic approaches for the long list of eEF2K-related human diseases and, more broadly, expand our understanding of translation elongation.

NIH Research Projects · FY 2025 · 2022-09

PROJECT ABSTRACT Heart disease is extremely prevalent, with about one in every four deaths (in the US) being attributed to heart disease. Early detection of cardiovascular events, especially before patients become symptomatic, has immense impact in preventive healthcare, reducing the morbidity and mortality associated with cardiovascular disease. Coronary artery calcification (CAC), a strong predictor for future cardiovascular events, is a component of atherosclerotic plaque buildup in the arteries that supply blood to the heart, leading to coronary artery disease (CAD). Identification of CAC is clinically important because it is used for cardiovascular risk and therapy decision making. Currently, CAC is quantified by computed tomography (CT), however, CT-based population screening is not widely utilized due to cost and radiation burden. Chest x-rays (CXR) are the most common medical imaging procedure and have higher availability than CT in low-resource settings, lower radiation dose, and higher patient throughput that could be used for screening purposes. Unfortunately, due to the lack of quantification in CXR, only qualitative descriptors are possible. The objective of this proposal is therefore to bring much-needed quantification to CXR, particularly for detecting and quantifying CAC by combining a new dual-layer x-ray detector and artificial-intelligence based image processing. The proposed dual-layer detector utilizes alloys of amorphous selenium (a-Se) that achieve favorable electro-optical properties (e.g., higher charge carrier mobilities and higher gain) compared to conventional a-Se based x-ray detectors. This technology has four major components: (1) a top layer direct convection a-Se alloys on an imaging backplane, (2) a bottom layer indirect conversion a-Se alloy with intrinsic gain on an imaging backplane coupled to a scintillator, (3) top panel and bottom panel integration into a dual-layer detector, and (4) a machine learning algorithm that enhances accuracy of the quantitative information from the dual-layer detector. The detector development leverages a mature platform from Varex Imaging, a leading manufacturer of x-ray detectors. We expect to show that the proposed system has higher spatial resolution images and higher sensitivity to detect small, high contrast features (calcifications) and to separate materials such as calcium from soft tissue. This approach will allow accurate quantification of predictive factors and will have immense impact in proactive healthcare, improving the clinical outcomes of patients, and reducing the number of deaths associated with cardiovascular disease. While our focus is on CAC, we expect this technology to broadly improve CXR for early detection of lung cancer, tuberculosis, and other diseases such as osteoporosis via quantification of bone mineral density.

NIH Research Projects · FY 2025 · 2022-09

Project Summary A major long-term goal of my laboratory is to determine molecular feedback mechanisms responsible for coordination between the cell division cycle and differentiation of embryonic stem cells (ESCs). ESCs have great therapeutic potential for regenerative medicine, because they can differentiate into any cell type and have unlimited self-renewal potential. This remarkable biological potential, known as pluripotency, is associated with a complex transcription network, an ultrafast cell division cycle that lacks typical checkpoints, and an atypical response to activation of the Mitogen-Activated Protein Kinase (MAPK) pathway. While a lot of progress has been made in characterizing the pluripotency transcription network, less is known about the cell division cycle of ESCs and its molecular links to the pluripotency transcription network. In addition, a critical yet poorly understood process is how ESCs use the MAPK pathway to control their self-renewal and differentiation. Our driving hypothesis is that the cell division cycle and pluripotency transcription network are linked through a bidirectional molecular feedback loop that is regulated by the MAPK activity. To test our hypothesis, we will: I. Characterize novel regulators of the MAPK pathway identified in our CRISPR screen in ESCs II. Determine functional substrate network of the MAPK kinases, Mek and Erk, in ESCs using chemical-genetic kinase engineering and quantitative phosphoproteomics. III. Determine mechanisms of pluripotency maintenance by the G1 cell cycle kinase (Cdk2) using direct labeling of the substrates by chemical-genetic engineering of Cdk2. To successfully complete proposed experiments, I have established collaborations with Professor Stanley Qi laboratory (Stanford University), Professor Alice Ting laboratory (Stanford University), Professor Seth Rubin laboratory (University of California, Santa Cruz) and Professor Boris Macek laboratory (University of Tubingen). By gaining detailed insight into the molecular mechanisms linking the cell division cycle and differentiation of ESCs, the outcome of this R35 proposal will provide novel strategies to address a key challenge in the field of regenerative medicine that is efficient and reproducible differentiation of ESCs for therapeutic purposes.

NIH Research Projects · FY 2025 · 2022-08

Project Summary/Abstract Within each cell, a robust molecular clock is established by transcription-translation feedback loops driven by the transcriptional activation complex CLOCK:BMAL1 and the repressors PER (period) and CRY (cryptochrome) that turn CLOCK:BMAL1 “off”. The molecular clock controls daily oscillations in the expression of over 40% of the genome to synchronize host physiology with the external environment. These oscillations are self-sustaining on the cellular level: circadian rhythms persist with similar timing even when all external cues are removed, but will align with external cues when present, a feature called entrainment. In mammals, induction of the circadian repressor PER is a universal first step in the entrainment. Microbes are ubiquitous in our environment and undergo daily fluctuations correlated with the 24-hour solar cycle, but it is not known how microbial exposure impacts circadian rhythms. This research will explore how microbial concentration affects cellular signaling cascades responsible for entrainment and will define novel innate regulators of the clock. In addition, these studies will leverage the genetic and experimental tractability of zebrafish to perform high throughput, real time kinetics of circadian responses in vivo. The ability to do whole body, non-invasive imaging in zebrafish has significant advantages over established murine models and is particularly advantageous for migratory populations such as immune cells. Furthermore, as a non-mammalian, cold-blooded vertebrate, the study of clocks in zebrafish also represents an incredible opportunity to bridge the evolutionary gap between the most well characterized animal systems: Drosophila (invertebrate) and mice (vertebrate, mammal). Together, this research will advance our fundamental understanding of vertebrate circadian clocks and how it integrates information about microbial stimulation to entrain cellular clocks at the molecular level.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY Our research is focused on the mechanisms that control the cell cycle of growth and division. The identification of proteins and pathways that regulate the decision to divide has led to inhibitors to treat cancer. However, the shortcomings of these inhibitors make it clear that there are still many molecular aspects of cell-cycle control that we do not understand, and further research is needed to innovate improved therapies. The cell cycle consists of two waves of gene expression to replicate DNA and undergo mitosis. We will study the structure, function, and regulation of proteins that control this cell- cycle dependent gene expression. These proteins include the transcription factors or TFs (e.g. E2F, B- Myb, FoxM1) whose activity stimulates transcription, proteins that negatively regulate the TFs (e.g. Rb, CycF, and the MuvB complex), and the kinases (e.g. Cdk, Aurora A, and Plk1) that phosphorylate and modulate TF activity. We will use an integrated approach that combines structural biology with cell- based assays to determine how these proteins interact with each other and chromatin to control transcription. These studies will provide new mechanistic insights regarding how the cell cycle is controlled and how transcription factors directly modulate gene expression through influencing chromatin structure.

NIH Research Projects · FY 2026 · 2022-08

Project Summary The incorporation of halogen atoms (F-, Cl-, Br-, I-) in small organic molecules plays a significant role in modulating their physical properties and biological activities while providing a synthetic handle for additional chemical modification. The regioselective and enantioselective installation of halogens in a strictly chemosynthetic manner is technically challenging and frequently utilizes toxic reagents and generates undesirable byproducts. In contrast, Nature has developed efficient enzymatic strategies to incorporate aqueous halide ions into organic scaffolds with negligible waste production. This proposal focuses on the exploration of a unique family of halogenating enzymes, specifically the bacterial site-specific vanadium dependent haloperoxidases (VHPOs), that use a coordinated vanadate ion (VO43-) and co-substrate hydrogen peroxide to oxidize aqueous halide ions and install them in a regio- and stereospecific manner on organic substrates. Despite their involvement in constructing multiple bioactive natural product scaffolds and catalyzing chemically diverse and useful reactions without additional cofactors or coenzymes, only a small fraction of the hundreds of site- specific VHPO homologs have been rigorously characterized. The exploration of this poorly defined chemical and biochemical space is what intellectually drives this proposal. Using interdisciplinary chemical, biochemical, and genomic techniques, we aim to better understand bacterial site-specific VHPO enzymology through three independent, yet interrelated objectives. The first involves the genomic identification and categorization of novel VHPO homologs available within publicly available repositories. Improved representation of microbial site- specific halogenases will permit us to correlate genomic sequences to biochemical reactivities with the ultimate intention of predicting chemistries directly from bioinformatic signatures. The second objective involves understanding the roles of uncharacterized VHPOs within bacterial secondary metabolism and chemical ecology. The majority of known bacterial site-specific homologs catalyze chemically unique reactions in natural product biosynthetic pathways; these biochemistries are critical for establishing the bioactivities of their cognate products. We propose that novel VHPO reactivities and diverse substrate scaffolds remain to be discovered, and that the use of homologous genes as biosynthetic ‘hooks’ will facilitate the genome-based identification of new secondary metabolites. Finally, we aim to define the structural determinants of halide and organic substrate specificity for synthetic applications, either within their native substrates or expanded to novel scaffolds. These objectives will simultaneously improve our understanding of VHPO halogenation enzymology at the substrate and macromolecular level and will facilitate biocatalytic efforts to apply these site-specific microbial halogenases towards chemically useful transformations.

NIH Research Projects · FY 2025 · 2022-07

ABSTRACT The UCSC Graduate Training Program in Genome Science combines cutting-edge computational biology training in a multidisciplinary biomedical science and engineering environment. Among other strengths, UC Santa Cruz is a world leader in computational biology, bioinformatics, genomic technologies and RNA biology. The goals of the program are to prepare trainees for advanced academic and industry careers in biomedical genomics. Our training program develops critical thinking skills, provides rigorous hands-on training in computer science, statistics, and biological sciences, and develops scientific communication skills. The proposed program will provide support for six trainees, each with a two-year appointment. Our trainee applicant pool consists of PhD students who have already joined UCSC’s interdisciplinary genomics and biomedical sciences community and completed three hands-on laboratory rotations during their first year of study, at least one of which must be conducted in the laboratory of a GS Program faculty member. Genome Science trainees participate in several innovative training activities. Each year, our trainees will organize and run a bootcamp for each year’s incoming PhD cohort of students who are accepted by the Dept. of Biomolecular Engineering. This 10-day activity is designed and implemented by the T32 trainees with feedback and guidance from faculty. The centerpiece of the bootcamp is an ambitious hands-on activity for the incoming grad cohort to tackle. Previous activities have included building a functional DropSeq single-cell RNA sequencing machine (2017), as well as genome sequencing and assembly of the banana slug – UCSC’s mascot (2019). Trainees receive training in labs of 26 faculty, including a winner of the MacArthur Award, members of the National Academy of Sciences, and a Nobel Laureate. Formal coursework for trainees includes graduate- level instruction in bioethics, programming, practical genomics, statistics, and scientific writing. These courses cover essential topics in rigor and reproducibility and responsible conduct of research. Working with the Graduate Advising Committee and their faculty mentors, trainees develop Individual Development Plans which may involve further coursework to address specific deficiencies or special interests. In coordination with the NHGRI DAP program, trainees will also serve as mentors to undergraduate students. Program success will be assessed via several quantitative metrics of trainee productivity, such as the timing and number of publications and success in applying for extramural research funds, as well as career outcomes that are tracked following the award of PhDs. These metrics will be assessed by the Genome Sciences Executive Committee. The program is interdisciplinary, including 26 program faculty from 8 departments. Most trainees and approximately half of program faculty are affiliated with the Biomolecular Engineering Department, as nearly all BME faculty have genomics-focused research programs.

NIH Research Projects · FY 2025 · 2022-05

PROJECT SUMMARY Respiratory syncytial virus (RSV) is the leading cause of severe lower respiratory tract disease in young children worldwide and is also a major cause of morbidity and some mortality in the elderly and immunocompromised. No approved RSV vaccine exists. Our goal is to utilize a structure-guided design approach to rationally engineer RSV G protein immunogens that induce robust and protective immunity against RSV. RSV G protein is one of two major immunogenic proteins on the RSV surface and has key roles in virus attachment to airway epithelial cells and virus modulation of innate immune defenses. RSV G protein is the target of neutralizing and protective antibodies. The G protein as a vaccine immunogen has been hampered by poor immunogenicity and by a paucity of structural information on its epitopes. In this proposal, we will test our central hypothesis that engineered multimeric RSV G immunogens that display protective conformational epitopes will elicit robust and protective RSV immunity. We will use an integrated approach to pursue four specific aims: (1) Use structural studies to define conserved RSV G protein epitopes recognized by protective antibodies, (2) Use structure-guided design to engineer multimeric RSV G protein immunogens, (3) Evaluate engineered RSV G protein immunogens for improved immunogenicity, and (4) Use a comprehensive immune analysis to evaluate RSV G protein CCD immunogens for balanced cytokine responses and protective antibodies made in response to vaccination. The proposed research will generate RSV G vaccine immunogens as candidates with demonstrated efficacy in preventing RSV infection and disease pathogenesis.

NIH Research Projects · FY 2026 · 2022-05

PROJECT SUMMARY The complexity of human splicing is daunting, yet intervention in splicing for treatment of diseases holds huge potential. Based on strong preliminary results, we propose three areas of investigation that leverage our group’s deep knowledge of splicing to address critical open questions, and to explore the potential for innovative engineering. The first area addresses the mechanism by which U2 snRNP captures the intron branchpoint early in spliceosome assembly, a step altered by recurrent cancer mutations and targeted in nature by antibiotic-producing bacteria. Using new reporters in which two branchpoints compete for recognition, we have identified a novel splicing fidelity mechanism we call “NO-BP decay,” in which U2 complexes that fail due to aberrant branchpoint selection are destroyed. We will characterize this process, applying a battery of candidate gene-based suppressor screens and biochemical tests in splicing extracts. The second area of investigation addresses how splicing is integrated with transcription and cell growth at the individual gene and cellular levels, an emerging area in need of innovation if splicing is to be successfully engineered. Preliminary results indicate that yeast cells have a limited capacity for splicing that creates competition for pre-mRNAs that is critical to cell function. We will measure both splicing capacity and the dynamics of competition, using RNA sequencing to develop a predictive model that explains how splicing is coordinated at a systems level. To understand the contribution of individual genes to this system we are applying synthetic biology approaches. We have engineered site-specific pauses of RNA polymerase II and shown that they alter splicing efficiency and alternative splicing, by unknown mechanism(s) that we will dissect. We will also explore in detail the role of splicing noise (stochastic variations in splicing output over time) on the ability of splicing to control stable homeostatic expression settings (as it does in many RNA binding protein genes) as well as to control a bistable switch (as it does in the Drosophila Sex lethal gene). These experiments will define the operational principles of simple splicing regulatory circuits. The third area of investigation is focused on the process of intron gain and its roles in eukaryotic gene creation and gene diversification. Our recent discovery that the spliceosome can convert the lariat intron to a true intron circle after splicing indicates that it can carry out reverse splicing reactions in vivo, raising questions about whether and how it might promote formation of new introns. We propose to test biochemical steps predicted to be necessary for spliceosome-mediated intron gain, and have already set up experiments to document intron gain in vivo. Given the fundamental conservation of the splicing machinery, this work promises to translate directly into new understanding of the mechanisms of gene regulation in eukaryotes, including humans. Defects in splicing are frequently recognized as contributors to disease, and interventions that address splicing defects are increasingly successful pathways to treatment.

NIH Research Projects · FY 2026 · 2022-05

ABSTRACT Amyloid β (Aβ) is a believed key toxic agent of Alzheimer’s Disease (AD). To develop AD therapeutics, an improved understanding of the mechanisms of Aβ toxicity is urgently needed. In the brain, Aβ is found mostly in extracellular deposits that may be taken up by neurons. The purpose of this proposal is to test the central hypothesis that neuronal Aβ uptake and toxicity are linked. Aβ forms diverse aggregates with varied neurotoxic profiles. Little is known about how structure and aggrega- tion state affect neuronal uptake and toxicity of Aβ. This makes it very difficult to devise strategies to block these pathogenic processes. Efforts to determine how conformation and aggregation state of Aβ affects its’ neuronal uptake and toxicity were hampered thus far by the lack of (a) methods to produce stable samples for structural analysis and (b) accurate tools to quantify neuronal uptake of different Aβ aggregates. Recent chirality-based approaches of the Raskatov lab have produced a set of stabilized oligomeric and fibrillary Aβ forms that will be used here as tools, with the goal to close this important knowledge gap. Proposed research is cross-disciplinary and collaborative: it includes structural collaborations with Dr. Eisenberg and Dr. Tycko; Dr. Glabe will consult on neurobiology experiments done in the Raskatov lab. The purpose of Aim 1 is to complete the structural elucidation of racemic Aβ fibrils by ssNMR, to then use those structural insights to devise smaller, more drug-like, oligomer-to-fibril converters, and to test the working hypothesis that oligomer-to-fibril conversion reduces Aβ uptake into neurons, thus suppressing its toxicity. This will be accomplished using C14-based radioquantitation tools in combination with various cell culture assays to measure both rapid and slow toxic actions of Aβ against neurons. Aim 2 will test the working hypothesis that the differences in toxicity between Aβ42-E22e and Aβ42-S26s are due to differences in their neuronal uptake, and will also test the alternative hypothesis that the peptides traffic to different sub-cellular sites, and that the differences in peptide toxicity are due to that. CryoEM structures of Aβ42-E22e and Aβ42-S26s stabilized oligomers will be sought, to identify the structural motifs responsible for their toxicity differences. Aim 3 will test the working hypothesis that the highly aggregation-prone, N-terminally truncated Aβ-related peptide p3 promotes oligomer-to-fibril conversion in Aβ, thus reducing Aβ uptake efficiency and making it less neurotoxic. Successful completion will yield a quantitative link between neuronal uptake and toxicity of different Aβ forms. It may yield the world’s first Aβ oligomer structures, as well as a structure of non-toxic Aβ fibrils. It may yield smaller, D-peptidic Aβ oligomer-to-fibril converters to be translated to novel AD therapeutics in the future, and it will also reveal how Aβ toxicity is suppressed by p3 addition. Finally, the proposed studies may uncover general structural insights on how protein aggregation affects neuronal uptake and toxicity.

NIH Research Projects · FY 2026 · 2022-05

The Institute for the Biology of Stem Cells (IBSC) at UCSC proposes a postdoctoral training program for 5 fellows each year (4 NICHD, 1 UCSC sponsored) that prepares postdoctoral fellows to take on leadership roles in professions that translate stem cell research into life-long health benefits. The trainees are sponsored for 2-year training periods that encompass rigorous research focused on normal and abnormal human development in a stem-cell context and individually tailored career development plans to prepare trainees for academic or biomedical industry jobs. The program was developed in response to research showing that structured mentoring and targeted career skill building improve career outcomes and increase the motivation of trainees to remain in science-focused careers. The core of the interdisciplinary training program consists of mentored research in laboratories of 24 faculty members (seasoned and new mentors) belonging to six departments: Molecular, Cell, and Developmental Biology; Biomolecular Engineering; Chemistry; Microbiology and Environmental Toxicology; Applied Mathematics; Electrical and Computer Engineering. All mentors accepted into this training program have well-funded stem cell research programs in NICHD focus areas and have strong scientific records. All mentors will receive regular mentoring training and new faculty will be paired with seasoned mentors. Trainees will also select co-mentors to support reaching their career goals. Trainees enter the program as a cohort and perform research in a mentor laboratory. In year 1, postdocs will also take part in formal training including grant writing, responsible conduct of research, and methods for enhancing reproducibility. In year 2, the program offers a number of additional training and practical opportunities that enhance leadership, mentoring, entrepreneurial, and communication skills. Participation is customized to each trainee’s career goals and will be guided by research mentors and co-mentors. The program is administered by an Internal Executive Committee and overseen by an External Advisory board. An External Selection Committee reviews and ranks program applicants for appointment to the program. An Entrepreneurship Committee will advise on program aspects designed for fellows with interest in industry careers. Program administration monitors training progress and program success through quarterly feedback, through annual program reviews by mentors and postdocs, and by documenting the career development of former trainees. This program is designed to improve the job satisfaction, productivity, retention, and career outcomes of postdoctoral fellows. The proposed training structure is supported enthusiastically by UCSC, as reflected in the substantial institutional support for this program.

NIH Research Projects · FY 2026 · 2022-04

PROJECT SUMMARY Complex decision-making often encompasses both cognitive and affective components. Cognitive and affective processes engage distinct but interacting systems in the brain, and both systems are effected by stress, a prevalent problem in modern society and a well-known risk factor for psychiatric disorders. Human brain imaging studies suggest a network of brain regions as the potential interface between cognitive and affective processing, particularly the medial prefrontal cortex (mPFC), the anterior insular cortex (aIC), and midbrain dopamine (DA) regions such as the ventral tegmental area (VTA). However, these studies lack causality tests at the neural circuit level. Comparative neuroanatomy and developmental genetics have identified in mice evolutionarily related mPFC, aIC, and DA regions. The structure and function of these regions are also disturbed by stress. These findings open the venue to use mouse as a model species for causal studies of the fundamental functions of these brain regions with precise circuit manipulation tools. Our overall goal is to elucidate the fronto-insular circuit mechanisms underlying cognitive-affective interactions during decision-making and the impact of stress on such mechanisms in mice. Our proposal is based on published literature and preliminary data showing: 1) mPFC, aIC and VTA are engaged in cognitive and affective decision-making; 2) mPFC and aIC are directly connected and both receive DA inputs from VTA; 3) stress affects mPFC and aIC neurons as well as DA release in these areas; 4) DA modulates mPFC and aIC activity and decision-making. By combining projection-specific viral labeling of neural circuits with in vivo imaging/electrophysiology and optogenetic/pharmacogenetic manipulations, we will test the central hypothesis that mPFC-aIC interaction is crucial for decision-making, which is disrupted by chronic stress but rescuable via DA modulation. Specifically, in Aim 1, we will determine the function of mPFC-aIC connections during decision-making in the attentional set-shifting test. In Aim 2, we will examine how stress affects mPFC-aIC connectivity and function. In Aim 3, we will define impact of stress on DA modulation of mPFC and aIC function, and explore the possibility to rescuing decision-making by selectively restore DA modulation in mPFC or aIC in stressed mice. The successful outcomes of this project will not only provide fundamental knowledge about the circuit mechanisms underlying higher brain functions, but also point out potential therapeutic targets for alleviating the detrimental effects of stress and psychiatric illnesses.

NIH Research Projects · FY 2025 · 2021-09

Dockstore - A Platform for Creating, Sharing, Publishing and Reproducing Computational Science For science to work analyses, tools and workflows must be reproducible; reproduction is essential to building consensus and confidence, and improving the state of the art. A large and growing set of scientific methods are computational in nature, and we can make these computational methods exactly reproducible. Dockstore is a repository for scientific tools and workflows that provides the means precisely to capture the code, parameters and operating system environment that are necessary. For tool users it makes it easy to find a given tool and then run it precisely as the author intended. It is possible to export a tool for use in a cloud platform, such as NHGRI AnVIL, with a few clicks, or similarly easily download it to an institutional compute environment, or even a laptop. For tool creators it provides an integrated system to publish versioned scientific tools with a citable Digital Object Identifier (DOI), linking to containers, existing code repositories and capturing vital parameter configuration information. Dockstore, previously funded by an R01 from NHGRI grant, has grown tremendously over the last four years. It now has thousands of users, hosts hundreds of tools and workflows, and is used as the methods for multiple major platforms and projects. Though not technically a renewal, this proposal would provide the core funding to continue the development and growth of Dockstore, which is a fully open source platform entirely reliant on grant funding. This proposal would grow Dockstore as a resource to make it a hub for broadly sharing computational science. The aims of the proposal cover foundational infrastructure development (Aim 1), support for sharing new, popular content types (Aim 2), the expansion of publishing features to make Dockstore more broadly useful (Aim 3), and a strong focus on training and outreach (Aim 4). The infrastructure development of Aim 1 will increase the scaling and robustness of Dockstore and support Dockstore as a secure, compliant resource, enabling it to safely host pre-publication tools and workflows working with managed access datasets. It will integrate Dockstore more deeply with secure cloud platforms, such as the NHGRI AnVIL. The expansion of content types by Aim 2 will, for example, make it possible to publish programming notebooks on Dockstore. Such artifacts are widely used but not yet robustly searchable by any scientific resource. Aim 3 will bring ORCID integration, allowing users to authenticate themselves and then sign and endorse their work, and to provide open peer review of published tools. Aim 4 will focus on growing and educating the user base via an expansion of online training material, webinars, in person workshops and collaborations with training for partner projects using Dockstore. We will also further develop means to continuously acquire user feedback.

NIH Research Projects · FY 2025 · 2021-09

ABSTRACT Center for Live Cell Genomics We will build new methodology and capacity for large-scale, long-term, inexpensive, modular, customizable, shared, Internet-of-Things-controlled, reproducible live cell culture and tissue-based experimental genomics disease models. Tissue models include traditional cell culture as well as organoid and primary tissue explants obtained from surgery or biopsy. Organoid factories supporting tissue growth and maintenance will be integrated with external and on-chip electro-optofluidic analytical modules to become part of an ecosystem that is modeled after open-source software. It will use commodity sensors, cameras, and computers linked in platforms flexibly designed using simple 3D printing, molding, etching and milling techniques potentially available at any institution. This will stimulate rapid innovation in experimental platforms for tissue culture. We will push this technology and use its best-in-class capabilities to make progress in neurodevelopment and pediatric cancer, addressing big questions. What genes contribute most importantly and specifically to human brain development? How do they go wrong in neurodevelopmental disease or brain injury? What specific molecular pathways are disrupted in individual pediatric cancer cases? How can we test pathway-specific treatments in a tissue model specific to each patient? Our education and outreach plans include a training program to develop a diverse and inclusive cohort of undergraduate students trained in genomic science through secondary school and community college outreach as well as coding workshops and research-based laboratory classes for UCSC undergraduates to develop core competencies. Participation in these activities serve as a basis for training graduate students and postdocs in inclusive pedagogy and mentorship. We are also developing a one-stop information hub to form an online community and to share our technology through immersive webinars and tutorials aimed at a broader audience with a range of expertise from the general public to scientists and clinicians at research institutions. Our work will enable significant advances in neuroscience and cancer research and education, stimulate a new open-source culture in cell biology and genomics, and democratize scientific and educational access beyond elite institutions, extending sharing projects like NHGRI ANvIL beyond data and code to include experiments and Internet-connected experimental platforms.